Sample dispensing device

ABSTRACT

A sample delivery device for delivering a particle-containing liquid comprises a sample chamber ( 12 ) provided in a syringe pump ( 10 ). The sample chamber ( 12 ) contains the liquid to be delivered. By moving a piston ( 14 ) in the direction indicated by an arrow ( 16 ) the sample liquid is, for example, fed through a tube ( 26 ) and a delivery opening ( 30 ) to wells of a titer plate. According to the invention, the sample chamber ( 12 ) is connected with a moving means ( 32 ), such as an electromotor, for preventing sedimentation of the particles contained in the liquid, and/or for preventing segregation of the sample liquid.

The invention relates to a sample delivery device for delivering aparticle-containing liquid.

Such a delivery device is, for example, a dispensing and/or pipettingmeans for delivering chemical and/or biological samples. For example, inhigh throughput screening a large number of wells in microtiter platesare filled with the aid of such sample delivery devices. In modern highthroughput screening plants microtiter plates having, for example, 384,1536, 2080 or more wells are used. Only very small sample volumes arefed to the individual wells. In particular, the volumes lie in the lowermicroliter and the supermicroliter range.

Suitable dispensing or pipetting means comprise, for example, amicropump having a piezoelectric element, with the aid of which pumpcorrespondingly small droplets for filling the wells can be produced.Further, known dispensing or pipetting means may comprise a syringe pumpwhose opening is, for example, connected via a tube with a high-speedvalve. For example, the piston of the syringe pump generates a pressurein the chamber of the syringe pump as well as in the adjoining tube, andby briefly opening the high-speed valve a correspondingly small volumeof liquid is delivered.

In particular because of the small volumes of the delivered sampleliquid the dwell time of the sample liquid in the sample chamber, i.e.for example in the chamber of the syringe pump, is relatively long.Depending on the composition of the sample liquid, this may lead tosegregation of the sample liquid. In particular during dispensing orpipetting of particle-containing liquids sedimentation of the particlesmay occur after a short period of time depending on the viscosity andsize of the particles. Tests have shown that frequently after as short aperiod as two minutes sedimentation of the particles occurs. Thisdestroys the homogeneity of the suspension. As a result, differentnumbers of particles are fed to the individual wells of the titer plate.A reproducible distribution of the particle-containing liquid is thus nolonger possible. An identical number of particles or an acceptable smalldeviation in the number of particles per well is thus not ensured. It istherefore not possible to dispense particle-containing liquids, forexample in high throughput screening plants, over a longer period oftime of possibly several hours. It must be particularly taken intoaccount that due to the small volumes in the individual wells even smalldeviations in the homogeneity of the liquid may lead to a considerablefalsification of the test results.

The term particle relates in particular also to cells. These are eitherindividual cells or cells which combine to form a particle.

Due to the sedimentation of particles the fluid systems, for exampletubes with small diameters, may become clogged. Such clogging can becaused by particles which deposited, for example, on the inner wall ofthe sample chamber and then came off.

Provision of agitating elements inside the sample chamber involves avery heavy technical expenditure due to the relatively small samplechambers used in particular in high throughput screening processes.Further, the use of agitating elements is of disadvantage in that theparticles, in particular cells, may become damaged. When syringe pumpsare employed, with the piston of the syringe pump being arrangeddirectly inside the sample chamber, provision of agitating elementsinside the sample chamber is further disadvantageous in that a deadvolume is created which cannot be emptied by the piston. Since highthroughput screening frequently involves the processing of veryexpensive sample liquids, provision of an agitating element in thesample chamber is not economical. Further, provision of additionalelements in the sample chamber has the drawback that the sterilizationprocess becomes more complex and thus more expensive. This is inparticular true with agitating elements which are in most casesdifficult to clean.

Since the sedimentation behaviour of the particles further depends onthe viscosity of the liquid, an adequate homogeneity of the liquid maybe attained by preventing viscosity for short periods of time. The useof viscosity-changing additives is however of disadvantage, inparticular with regard to cells, in that the cells may become damaged oraffected. Further, although the use of viscosity-changing additives mayreduce the sedimentation tendency of the particles, it does noteliminate said tendency. In particular in high throughput screeningplants in which sample liquids are fed from the sample chamber to theindividual wells of the titer plates over a period of several hours, theemployment of viscosity-changing additives is not possible due to thelong time periods involved.

It is an object of the invention to provide a sample delivery device fordelivering particle-containing liquids, with the aid of which thehomogeneity of the liquid can be created and/or maintained.

According to the invention, this object is achieved with the features ofclaim 1.

The sample delivery device according to the invention, which is, forexample, a dispensing or pipetting means, comprises a sample chamber forcontaining the liquid to be delivered and a feed means for feeding theliquid towards a delivery opening. For maintaining the homogeneity ofvery small liquid quantities in the sample chamber, which are to bedelivered, for example, to wells of a titer plate, the sample deliverydevice comprises, according to the invention, a moving device for movingthe sample chamber. By moving the sample chamber and thus the liquid inthe sample chamber, for example by shaking, turning or reciprocating thesample chamber, the homogeneity of the sample liquid is maintainedand/or created. The sample delivery device according to the invention isthus in particular suitable for delivering, i.e. dispensing orpipetting, a particle-containing, in particular cell-containing, liquid.

By moving the sample chamber the homogeneity of a suspension in thesample chamber is permanently maintained. It is thus in particularpossible to keep a sample liquid homogeneous over a longer period oftime of, for example, several hours and process said liquid, for exampleby high throughput screening. In particular in the case of very smallsample quantities of only a few microliters or even a few nanoliters theparticle number in the individual wells can be kept constant with, forexample, small deviations, upon filling the individual wells inparticular in the high throughput screening process. Since the movingmeans according to the invention does not affect the sample liquid aswould be the case if agitating means or the like were used, inparticular the vitality of the cells is not affected due to the factthat the homogeneity is maintained with the aid of the moving meansaccording to the invention. Further, the device according to theinvention prevents sedimentation. Consequently, the fluid system cannotbecome clogged or contaminated by deposits. Further, an unintentionalmixing of sediments with a new liquid is thus prevented.

The sample chamber can, for example, be a liquid reservoir which ispreferably connected via a tube or a duct with a feed means, such as amicropump. The micropump comprises a corresponding delivery openingthrough which the liquid is delivered into a well or any other receivingmeans. The moving means according to the invention is connected with thereservoir and sets the reservoir together with the liquid in motion.

Preferably, the sample chamber is the chamber of a syringe pump in whichthe piston of the syringe pump is guided. In this case the moving meansis connected with the overall syringe pump such that the overall syringepump is moved to maintain and/or create the homogeneity of the liquid inthe sample chamber, i.e. the chamber of the syringe pump. Provision ofsuch a piston pump as feed means offers the advantage that the samplereservoir is arranged directly inside the pump. Since according theinvention no means such as agitators or the like are arranged inside thesample chamber, the liquid can also be moved from outside with the aidof the moving means such that the sample chamber can be completelyemptied by the piston of the piston pump. A dead volume is not createdinside the sample chamber. This ensures economical use of the expensivesample liquids. Further, mixing of two liquids, which are processed oneafter the other, is prevented. Moreover, cleaning of such a piston pumpwithout any additional agitating means or the like is considerablyeasier such that, for example, good sterilization conditions can beguaranteed.

The feed means of the sample delivery device, which is in particular asyringe pump, is, for example, connected via a fluid segment, such as atube, with a delivery means comprising a delivery opening. The deliveryopening may, for example, be a tube end. Preferably, the deliveryopening is provided in a tip or the like which is connected with thetube and may be exchangeable.

The feed means, in particular the syringe pump, is preferably connectedvia a holder with the moving means. The moving means may, for example,be an electromotor, in particular a step motor, with the aid of whichturning of the sample chamber is realized. In particular, turning iseffected about the transverse axis of the piston pump, in particular thesyringe pump. In this connection, a syringe pump comprises anessentially cylindrical cavity serving as sample chamber. In this cavitya piston is guided, wherein movement of the piston in longitudinaldirection of the cylindrical cavity causes liquid to be delivered via apump outlet which is normally arranged opposite the piston.

To improve the homogeneities of the liquids, turning is not effectedcontinuously in one direction but the sense of turning is changed, i.e.the sample chamber is reciprocated. Preferably, the sense of turning ischanged at an angle of turning between 180 and 360 degrees. Theresulting accelerations of the liquid and the particles, in particularthe cells, contained in the liquid ensure thorough mixing andmaintaining of the homogeneity.

Hereunder the invention is explained in detail on the basis of apreferred embodiment with reference to the accompanying drawings inwhich:

FIG. 1 shows a schematic side view, partly in section, of the deviceaccording to the invention,

FIG. 2 shows a schematic front view of the device as seen in thedirection of arrow II of FIG. 1,

FIG. 3 shows a diagram representing the distribution of cells in a titerplate in the case of dispensing without the moving means according tothe invention, and

FIG. 4 shows a diagram representing the distribution of cells in a titerplate in the case of dispensing with the moving means according to theinvention.

The sample delivery device schematically shown in FIGS. 1 and 2, whichis particularly suitable for delivering very small quantities ofparticle-containing liquids, i.e. for example into individual wells oftiter plates, comprises a syringe pump 10 serving as a feed means forfeeding a liquid. The liquid containing in particular particles islocated in a sample chamber 12 of the syringe pump. The sample chamber12 is, for example, of cylindrical configuration. In the sample chamber12, which, in the embodiment shown, is the piston space of the syringepump 10, a piston 14 is arranged which is capable of being displaced inthe piston space 12 in the direction indicated by an arrow 16. When thepiston 14 is introduced into the piston space or sample chamber 12 inlongitudinal direction of the housing 18 defining the cylindrical samplechamber, sample liquid is delivered from the sample chamber through anopening 22 provided in a bottom 20 of the housing 18. The opening 22 isarranged in the bottom 20 opposite the piston 14 and is generallyconcentrically with the piston 14. At the opening 22 a hub 24 isprovided, to which, in the embodiment shown, a tube 26 is attached. Thetube 26 is connected with a delivery tip 28. The delivery tip 28 may beexchangeable and comprises a delivery opening 30 through which theliquid is delivered towards the individual wells of a titer plate.

Additionally, a high-speed valve can be provided in the tube 26 or inthe delivery tip 28. The high-speed valve can precisely control thedelivery of the sample liquid. Further, provision of a high-speed valveallows the piston 14 to be continuously pushed into the piston space 12,and the drop-by-drop delivery of the sample liquid to be exclusivelycontrolled via the valve.

It is further possible to connect the sample chamber or piston space 12with a reservoir which contains a larger quantity of sample liquid. Thiscan, for example, be realized via an additional opening in the housing18 or via a tube 26 branch provided with a valve. By correspondinglyswitching the valve, the syringe pump can be drawn up by withdrawing thepiston 14, thus drawing in sample liquid from the reservoir. Thisreservoir may be connected, in addition to the syringe pump 10, with amoving means for moving the liquid and thus maintaining the homogeneityof the liquid.

The volume of the pump chamber 12 is, in particular when the sampledelivery device according to the invention is employed, a great deallarger than the individual volume of the very small sample liquidquantities delivered into the individual wells. In particular, thevolume of the pump chamber 12 is ten times larger, preferably at least ahundred times larger, that the delivered individual volumes.

To maintain the homogeneity of the sample liquid in the pump chamber 12the syringe pump 10 is, according to the invention, connected with amoving means 32 (FIG. 2), such as an electromotor. Preferably, theelectromotor 32 is a step motor. The syringe pump 10 is connected via aholding or clamping device 34 and a shaft 36 with the motor 32. With theaid of the motor 32 the syringe pump 10 can be preferably reciprocatedin the direction indicated by an arrow 38. This results in turning ofthe syringe pump 10 along with the sample chamber 12 about a transverseaxis 40 of the syringe pump 10. In particular during the reciprocatingmovement accelerations occur which cause the particles, in particularthe cells, in the sample liquid to be homogeneously distributed orremaining homogeneously distributed. In the case of liquids which do notcontain any particles the moving means 32 according to the inventionprevents the liquids from segregating.

The inventive movement of the syringe pump and connection of the syringepump via a tube with a delivery means 28 or the like allow the samplechamber 12 to be permanently kept in motion, irrespective of whether ornot liquid is delivered during the movement. Movement of the samplechamber 12 and thus the liquid contained in the sample chamber need thusnot be interrupted for the purpose of delivering liquid. Preferably, thesyringe pump 10 is already in motion when the sample chamber 12 is beingfilled with sample liquid. In the case of a pipetting device filling canbe carried out through the tube 26 by drawing sample liquid through theopening 30 and the tube 26 into the sample chamber. In the case of adispensing means the sample chamber 12 is connected with an additionalreservoir such that the sample liquid is drawn from the reservoir intothe sample chamber 12 and is then delivered through the tube 26 and theopening 30 into the wells or the like. It is further possible to movethe syringe pump 10 over a given period of time of, for example one tothree minutes, after filling of the sample chamber 12 and prior todelivering the sample liquid through the delivery opening 30 to ensurethat the liquid in the sample chamber 12 has a high homogeneity beforeinitial delivery of a small quantity of sample liquid. This guaranteesthat even at the beginning of sample liquid delivery no variations inthe cell number or the like occur.

The diagram shown in FIG. 3 represents a test result with regard to thenumber of cells and the portion of dead cells, wherein dispensing of thesample liquid was carried out using a sample delivery device without amoving means. The sample liquid was thus not moved in the sample chamber12 during the delivery process. In the diagram, the cell numbers areplotted as grey bars against a row of a titer plate. The individualwells of the rows of the titer plates were filled one after the other.Filling was effected with the aid of the dispensing system PreSys of theCartesian company. During the filling process the piston of the syringepump was continuously moved. Filling of the individual wells was carriedout by correspondingly actuating a high-speed valve provided in the tubewhich connects the syringe pump with the outlet means. For determiningthe overall cell number per row of the titer plate, the cells were dyedprior to the dispensing process with a vital dye combining with proteinsin the cytoplasm. CMFDA (chloromethylfluorescein diacetate) was used asdye. By charging ethidium homodimer into the individual wells the cellcore of killed cells was dyed either after the dispensing process.Readout of the titer plate was effected using an image system forfluorescent samples. In this connection, the two dyes were excited bylight with a wavelength of 480 nm. The emission wavelength for CMFDA is510 nm and the emission wavelength for ethidium homodimer is 680 nm.

As can be seen from the Figure, the cell number considerably variesbetween the individual rows of the titer plate. The variation limitsrange between approximately eighty and one hundred and forty cells perrow. Such high variations in the cell numbers lead to a strongfalsification of the test results. The dead portion also varies stronglyacross the individual rows of the titer plate.

Compared to this, the same test as explained with reference to FIG. 3was carried out, as shown in FIG. 4, wherein however the syringe pump 10was moved by the moving means according to the invention during theoverall dispensing process. As can be seen from FIG. 4, the cell numberis subject to a considerably smaller variation. The percentage of thedead portion, too, is subject to a substantially smaller variation andthus negligible. Further, it turned out that the initial concentrationof the dispensed sample liquid and the measured number of cells were thesame.

Further, in both FIGS. 3 and 4 the standard deviations are plotted.These deviations, too, are considerably smaller when the syringe pump ismoved (FIG. 4).

1. Cell suspension delivery device for delivering a cell-containing liquid, in particular in very small quantities, comprising a sample chamber for containing the liquid to be delivered, and a feed means connected with the sample chamber for feeding the liquid towards the delivery opening, and a moving means connected with the sample chamber for moving the liquid in the sample chamber wherein the moving means causes the sample chamber to be turned about the transverse axis of the feed means.
 2. Cell suspension delivery device according to claim 1, characterized in that a piston pump, in particular a syringe pump, is provided as feed means.
 3. Cell suspension delivery device according to claim 2, characterized in that a piston space in which a piston of the piston pump is arranged serves as sample chamber.
 4. Cell suspension delivery device according to claim 1, characterized by a change of the sense of turning.
 5. Cell suspension delivery device according to claim 1, characterized in that the volume of the sample chamber is a great deal larder than the individual volume of the delivered very small quantities.
 6. Cell suspension delivery method for delivering a cell-containing liquid in particular in very small quantities the method comprising the following steps: maintaining the homogeneity of the cell suspension contained in the sample chamber by turning the sample chamber about a transverse axis of a feed means connected with the sample chamber, and feeding the liquid with the aid of the feed means towards a delivery opening.
 7. Cell suspension delivery device according to one of claim 6, wherein maintaining of the homogeneity is effected exclusively by turning about the transverse axis.
 8. Cell suspension delivery method according to claim 6, wherein the sense of turning of the sample chamber is changed.
 9. Cell suspension delivery method according to claim 6, wherein the sample chamber is reciprocated.
 10. Use of a sample delivery device comprising a sample chamber for containing a liquid to be delivered, a feed means connected with the sample chamber for feeding the liquid towards the delivery opening, and a moving means connected with the sample chamber for moving the liquid in the sample chamber, in particular a cell suspension delivery device according to claim 1, for delivering a cell-containing liquid, in particular by applying the method according to claim
 6. 